Abstract A universal influenza vaccine that protects against all strains of influenza is a high priority. The Centers for Disease Control & Prevention (CDC) estimate the economic impact of seasonal influenza in the United States ranges from $10 to $16 billion and pandemic influenza ranges from $71.3 to $166.5 billion. The proposed research directly addresses the limitations of both pandemic and seasonal flu vaccines. Versatope has developed a unique influenza M2e antigen construct that is potentially effective against several strains of influenza in mouse and ferret animal models. It is expressed in Escherichia coli and derived from recombinant outer membrane vesicles (rOMVs) or M2e-rOMVs. The ferret study showed higher levels of antigen- specific antibodies and lower viral loads of pandemic H1N1 influenza following prime/boost administration of M2e-rOMVs compared to a commercially available influenza vaccine. The OMV vaccine delivery is innovative because our E. coli production strain has been genetically engineered to detoxify lipopolysaccharide (LPS) more than 1000-fold (they do not require chemical extraction of LPS to detoxify the final product) and to increase OMV formation more than 30-fold compared to the parental probiotic strain of BSL1 bacteria. Since it is known that influenza undergoes antigenic variation under immunologic selection, the M2 ectodomain may not be a sustainable vaccine candidate for long-term preventative applications in humans. Although our current M2e-rOMV vaccine candidate contains diverse strain sequences from one target antigen, we propose to develop a multi-antigen influenza vaccine candidate and to demonstrate improved protection against heterologous challenge using the multivalent influenza rOMV compared to the M2e-rOMV in the mouse model. We expect that our approach using multi-antigen (conserved domains from representative hemagglutinin, neuraminidase and nucleoprotein together with M2 ectodomains) influenza construct will yield stable rOMVs and provide with protection against multiple influenza strains. OMVs are ideally suited for a multi- valent vaccine because recombinant proteins can be expressed as fusion proteins and/or independently targeted to the lumen, the membrane, or the surface of OMVs. Our proposed research program is primarily translational, the outcome of which will guide the path toward a viable single-dose vaccine for pandemic influenza A. The development of this new multivalent influenza-rOMV will enable large-scale production suitable for non-clinical development and toxicology, clinical studies, and commercial development. We will also identify the minimum dose required for immunogenicity for future safety/toxicity studies. These rOMVs represent a potentially safe and simple subunit vaccine delivery platform that will increase the range of protection against multiple strains of pandemic and seasonal influenza and reduce the overall economic impact.